Genetic markers of the risk of coronary heart disease and coronary artery thrombosis developing in the Kazakh population

Background: Surgical methods such as coronary artery bypass grafting and percutaneous coronary interventions (PCI) are widely used along with traditional conservative therapy in the treatment of coronary artery disease. The disease outcome directly depends on timely diagnosis and treatment. A significant role in predicting the effectiveness of treatment is given to personification of treatment and management of the patient. In this case, the determining component is its individual genetic status. Methods: The study groups included persons of Kazakh nationality which identify themselves, their biological parents, and biological grandparents on the maternal and paternal side as Kazakh. Research groups included 108 people at the age from 45 to 65 years of both sexes. Blood samples genotyping was carried out by PCR using highly specific TaqMan samples. Thermo Fisher cloud application was used for genotypes determining on the base of an automatic algorithm. Results: The article presents the results of the evaluation of gene polymorphisms associated with coronary artery restenosis in a population of Kazakh nationality. 3 SNPs were determined when searching for an association with stenting due to coronary artery thrombosis: rs7543130 (p=0.009324), rs6785930 (p=0.016858), rs7819412 (p=0.061325). Conclusion: Four polymorphisms associated with the risk of developing coronary heart disease were revealed during the study of polymorphisms among the people of the Kazakh population. Three SNPs were determined when searching for an association with stenting due to coronary artery thrombosis. It should be noted that the Bonferonni correction for multiple comparisons did not reveal significant polymorphisms associated with coronary artery disease, which requires further research with more quantity of samples.

Coronary heart disease (CHD) is one of the main causes of cardiovascular mortality (1). Despite significant progress in the development and application of modern methods of diagnosis and treatment, diseases of the circulatory system are the most important problem in terms of mortality in the Republic of Kazakhstan (2). According to WHO experts, over the past 10 years there has been a tendency to increase the number of diseases of the circulatory system by 1.3 times (from 1984.4 per 100 thousand population in 2003 to 2523.0 per 100 thousand population in 2012) (3). In general, diseases of the cardiovascular system in Kazakhstan are the cause of almost 1/3 of all deaths. In the structure of mortality from diseases of the circulatory system 34% are patients with coronary heart disease, of which more than 30% are persons of active working age (18-65 years old) (4,5).

Taizhanova D, et al.
Surgical methods such as coronary artery bypass grafting and percutaneous coronary interventions (PCI) are widely used along with traditional conservative therapy in the treatment of coronary artery disease. The disease outcome directly depends on timely diagnosis and treatment. A significant role in predicting the effectiveness of treatment is given to personification of treatment and management of the patient. In this case, the determining component is its individual genetic status. To evaluate the genetic polymorphisms determining the predisposition to coronary heart disease in individuals of the Kazakh population and the predisposition to subsequent coronary artery thrombosis, that necessitated stenting.

Methods
The study groups included persons of Kazakh nationality which identify themselves, their biological parents, and biological grandparents on the maternal and paternal side as Kazakh. Permission of the Committee on Bioethics of KSMU No. 305 dated May 19, 2017, was received for conducting research. The subjects were divided into the following groups: the I st group -58 people at the age from 45 to 65 years of both sexes. Inclusion criteria: patients with a diagnosis of coronary heart disease receiving traditional medication based on the recommendation of the European Society of Cardiology (ESC). The II nd group -58 people aged from 45 to 65 years of both sexes. Inclusion criteria: patients with diagnosed coronary artery disease after stenting due to coronary artery thrombosis and lack of restenosis during the year (ESC). The control group included 50 people at the age from 45 to 65 years of both sexes. Inclusion criteria: practically healthy persons (not registered in the dispensary). A survey of patients was conducted to identify risk factors for thrombosis, gender and anthropometric characteristics (age, height, body mass index, waist circumference) were assessed, a detailed analysis of the provided medical documentation of patients was carried out -discharge summaries (based on the written consent of the patients). The study material was the blood of patients and conditionally healthy people. Blood sampling was carried out as standard in the morning, on an empty stomach. 2 ml of whole blood was taken into a vacuum container with the anticoagulant EDTA for molecular genetic studies. Blood for molecular genetic studies was collected once. Total whole blood DNA was extradited using the commercial GeneJET Genomic DNA Purification Kit (Thermo Scientific). The concentration and degree of purification of the isolated DNA were monitored spectrophotometrically using a P330 nanospectrophotometer (Implen) and a LabChip chip electrophoresis system. Genotyping of samples was carried out using QuantStudio TM 12K Flex Real-Time PCR (Applied Biosystems) technology using genetic panel (55 polymorphisms in each). The analysis of PCR results (QuantStudio 12K) was carried out in the ThermoFisher cloud service (https://apps.thermofisher.com). The results of each PCR reaction were presented in a 2-dimensional space to visually assess the distribution into pools (figure 1). After automatic annotation and visual inspection, genotypes were determined for each sample (patient). Figure 1 -Twodimensional distribution of genotyping results The following indices were determined for each polymorphism included in the genotyping panel: major and minor alleles, an indicator of minor allele frequency (MAFminor allele frequency), relative values for alleles and genotypes, and also an indicator of p-value when calculating the Hardy -Weinberg equilibrium (HWE -Hardy -Weinberg equilibrium) by groups. All SNPs corresponded to the Hardy-Weinberg equilibrium. Statistical analysis was carried out in the programs R statistics (Compare Groups and rstatix packages).

Results
Such questionnaire data as alcohol consumption (p<0.001), smoking (p=0.004), consumption of animal fat (p=0.008) have significant differences in the groups and, of course, lead to the manifestation or complication of cardiovascular pathology. It is worth noting that the data demonstrate a change in the lifestyle of patients after the manifestation of pathology. Such a risk factor as heredity is well traced in the group with coronary heart disease (88%, p<0.001), while patients with stenosis noted less hereditary component (47.5%, p<0.001), which may indicate a different etiopathogenetic mechanism of pathology. Concentrations of HDL (p<0.001), LDL (p<0.001) and triglycerides (p=0.017) had significant differences in the groups, as well as coagulogram parameters (Fibrinogen (p=0.003), PTI (p<0.001), prothrombin time (p<0.001)). Such indicators can be explained by the use of hypolipidemic and anticoagulant therapy. The data is presented in table 1.
A genetic panel for 55 polymorphisms was developed during the determining of the genetic markers associated with the risk of CHD developing in patients with stenting (second group). Information on the nucleotide sequence of the selected polymorphisms, their location in the chromosomes, genes, and genetic domain are presented in table 2. Polymorphisms associated with the risk of coronary heart disease developing are most often located on 1, 10, 7 chromosomes. The CYP2C19 gene has 7 polymorphisms in structure included in the panel. MED12L and PEAR1 genes have 3 SNPs each. The presented polymorphisms are most often located in proteincoding regions and are associated with variants of introns. The results of the statistical analysis of genotyping are presented in table 3. The results of the analysis allow us to conclude that despite the fact that all four polymorphisms have a p-value of less than 0.05, 95% CI calculated for OR indicates that only 2 polymorphisms (rs762551 and rs6785930) minor allele reduce the risk of re-stenting by 1.85 and 1.78 times, respectively.
All polymorphisms are different, not overlapping between groups. The assessment of the association of genetic polymorphisms in the «Control vs Stenting», «Control vs CHD» groups was carried out in accordance with the casecontrol design based on the generalized linear model (GLM) assuming a log-additive inheritance model. 4 polymorphisms associated with the risk of CHD were identified when comparing the results of genotyping in groups of patients with coronary artery disease with control: rs762551 (p=0.019), rs12976411 (p=0.011), rs2242480 (p=0.017) and rs4977574 (p=0.02). The assumptions were applied (p-value is higher, but close to 5%) for the analysis of the results of patients with installed stent. 3 SNPs associated with stenting were identified: rs7543130 (p=0.009), rs6785930 (p=0.016), rs7819412 (p=0.061).

Discussion
The rs762551 polymorphism is in the CYP1A2 gene, the product of which is involved in many metabolic reactions, including methylation (R-HSA-156581), metabolic oxidation (R-HSA-211859), fatty acid metabolism (R-HSA-8978868), protectin biosynthesis (R-HSA-9018681) and much more (6). At the same time, the rs6785930 polymorphism is in the P2RY12 gene, the product of which is involved in processes related to the transmission/transfer of signals via the P2Y receptor (R-HSA-417957, R-HSA-392170) and platelet activation and aggregation (R-HSA-76002) (7,8).
It should be noted that the Bonferonni correction for multiple comparisons did not reveal significant polymorphisms associated with phenotypes (CHD, stenting, restenosis after stenting). At the same time, without taking into account the Bonferonni correction, 3 SNPs associated with stenting and 4 SNPs with coronary artery disease were detected with a small assumption (p-value is higher, but close to 5%).  In conclusion, four polymorphisms associated with the risk of developing coronary heart disease were revealed during the study of polymorphisms among the people of the Kazakh population: rs762551 (p=0.019), rs12976411 (p=0.011), rs2242480 (p=0.017) and rs4977574 (p=0.02). Three SNPs were determined when searching for an association with stenting due to coronary artery thrombosis: rs7543130 (p=0.009), rs6785930 (p=0.016), rs7819412 (p=0.061). It should be noted that the Bonferonni correction for multiple comparisons did not reveal significant polymorphisms associated with coronary artery disease, which requires further research with more quantity of samples.